QC Report


general
Report generated at2024-11-08 20:47:40
TitleHumanliver_ATAC-seq
DescriptionHumanliver_hg38_ATAC-seq_encode_pipeline_Mary
Pipeline versionv2.2.3
Pipeline typeatac
Genomehg38
Alignerbowtie2
Sequencing endedness{'rep1': {'paired_end': True}, 'rep2': {'paired_end': True}}
Peak callermacs2

Alignment quality metrics


SAMstat (raw unfiltered BAM)

rep1rep2
Total Reads193675982300654650
Total Reads (QC-failed)00
Duplicate Reads00
Duplicate Reads (QC-failed)00
Mapped Reads191593429296451244
Mapped Reads (QC-failed)00
% Mapped Reads98.998.6
Paired Reads193675982300654650
Paired Reads (QC-failed)00
Read196837991150327325
Read1 (QC-failed)00
Read296837991150327325
Read2 (QC-failed)00
Properly Paired Reads189518268293213598
Properly Paired Reads (QC-failed)00
% Properly Paired Reads97.8999999999999997.5
With itself190875260294977500
With itself (QC-failed)00
Singletons7181691473744
Singletons (QC-failed)00
% Singleton0.40.5
Diff. Chroms2745444867
Diff. Chroms (QC-failed)00

Marking duplicates (filtered BAM)

rep1rep2
Unpaired Reads00
Paired Reads78364751126687267
Unmapped Reads00
Unpaired Duplicate Reads00
Paired Duplicate Reads2924039924698587
Paired Optical Duplicate Reads00
% Duplicate Reads37.313219.4957

Filtered with samtools flag 1804 (samtools view -F 1804):


Fraction of mitochondrial reads (unfiltered BAM)

rep1rep2
Rn = Number of Non-mitochondrial Reads128802940257568789
Rm = Number of Mitochondrial Reads6879958242695616
Rm/(Rn+Rm) = Frac. of mitochondrial reads0.348171578498375660.14219339784880594

SAMstat (filtered/deduped BAM)

rep1rep2
Total Reads95205822201514074
Total Reads (QC-failed)00
Duplicate Reads00
Duplicate Reads (QC-failed)00
Mapped Reads95205822201514074
Mapped Reads (QC-failed)00
% Mapped Reads100.0100.0
Paired Reads95205822201514074
Paired Reads (QC-failed)00
Read147602911100757037
Read1 (QC-failed)00
Read247602911100757037
Read2 (QC-failed)00
Properly Paired Reads95205822201514074
Properly Paired Reads (QC-failed)00
% Properly Paired Reads100.0100.0
With itself95205822201514074
With itself (QC-failed)00
Singletons00
Singletons (QC-failed)00
% Singleton0.00.0
Diff. Chroms00
Diff. Chroms (QC-failed)00

Filtered and duplicates are removed. Subsampling with atac.subsample_reads is not done in alignment steps. Nodup BAM is converted into a BED type (TAGALIGN) later and then TAGALIGN is subsampled with such parameter in the peak-calling step.

Fragment length statistics (filtered/deduped BAM)

rep1rep2
Fraction of reads in NFR0.58199358976415590.5207237190578763
Fraction of reads in NFR (QC pass)TrueTrue
Fraction of reads in NFR (QC reason)OKOK
NFR / mono-nuc reads2.2114791471100981.8753482820014797
NFR / mono-nuc reads (QC pass)FalseFalse
NFR / mono-nuc reads (QC reason)out of range [2.5, inf]out of range [2.5, inf]
Presence of NFR peakTrueTrue
Presence of Mono-Nuc peakTrueTrue
Presence of Di-Nuc peakTrueTrue

rep1
rep1
rep2
rep2

Open chromatin assays show distinct fragment length enrichments, as the cut sites are only in open chromatin and not in nucleosomes. As such, peaks representing different n-nucleosomal (ex mono-nucleosomal, di-nucleosomal) fragment lengths will arise. Good libraries will show these peaks in a fragment length distribution and will show specific peak ratios.



Sequence quality metrics (filtered/deduped BAM)

rep1
rep1
rep2
rep2

Open chromatin assays are known to have significant GC bias. Please take this into consideration as necessary.


Annotated genomic region enrichment

rep1rep2
Fraction of Reads in universal DHS regions0.37661336509441620.3920419275529113
Fraction of Reads in blacklist regions0.00192703551259711830.0015658807036971522
Fraction of Reads in promoter regions0.156655094055067330.1631528723894491
Fraction of Reads in enhancer regions0.31130377719967590.31495788725903084

Signal to noise can be assessed by considering whether reads are falling into known open regions (such as DHS regions) or not. A high fraction of reads should fall into the universal (across cell type) DHS set. A small fraction should fall into the blacklist regions. A high set (though not all) should fall into the promoter regions. A high set (though not all) should fall into the enhancer regions. The promoter regions should not take up all reads, as it is known that there is a bias for promoters in open chromatin assays.


Library complexity quality metrics


Library complexity (filtered non-mito BAM)

rep1rep2
Total Fragments54025915112851930
Distinct Fragments47790957101513762
Positions with Two Read49687969056123
NRF = Distinct/Total0.8845930.899531
PBC1 = OneRead/Distinct0.8843360.900864
PBC2 = OneRead/TwoRead8.50573210.098147

Mitochondrial reads are filtered out by default. The non-redundant fraction (NRF) is the fraction of non-redundant mapped reads in a dataset; it is the ratio between the number of positions in the genome that uniquely mapped reads map to and the total number of uniquely mappable reads. The NRF should be > 0.8. The PBC1 is the ratio of genomic locations with EXACTLY one read pair over the genomic locations with AT LEAST one read pair. PBC1 is the primary measure, and the PBC1 should be close to 1. Provisionally 0-0.5 is severe bottlenecking, 0.5-0.8 is moderate bottlenecking, 0.8-0.9 is mild bottlenecking, and 0.9-1.0 is no bottlenecking. The PBC2 is the ratio of genomic locations with EXACTLY one read pair over the genomic locations with EXACTLY two read pairs. The PBC2 should be significantly greater than 1.


Fragment: read for a single-ended dataset, pair of reads for a paired-ended dataset
NRF: non redundant fraction
PBC1: PCR Bottleneck coefficient 1
PBC2: PCR Bottleneck coefficient 2
PBC1 is the primary measure. Provisionally


Replication quality metrics


IDR (Irreproducible Discovery Rate) plots

rep1_vs_rep2
rep1_vs_rep2
rep1-pr1_vs_rep1-pr2
rep1-pr1_vs_rep1-pr2
rep2-pr1_vs_rep2-pr2
rep2-pr1_vs_rep2-pr2
pooled-pr1_vs_pooled-pr2
pooled-pr1_vs_pooled-pr2

Reproducibility QC and peak detection statistics

overlapidr
Nt223290128816
N1188490103876
N2229809148380
Np243527159958
N optimal243527159958
N conservative223290128816
Optimal Setpooled-pr1_vs_pooled-pr2pooled-pr1_vs_pooled-pr2
Conservative Setrep1_vs_rep2rep1_vs_rep2
Rescue Ratio1.09063101795870841.24175568252391
Self Consistency Ratio1.21921056819990461.4284339019600292
Reproducibility Testpasspass

Reproducibility QC


Number of raw peaks

rep1rep2
Number of peaks299245299390

The number of peaks is capped at 300000
Peaks are called from macs2 with p-val threshold 0.01

Peak calling statistics


Peak region size

rep1rep2idr_optoverlap_opt
Min size150.0150.0150.0150.0
25 percentile211.0241.0466.0333.0
50 percentile (median)334.0409.0715.0547.0
75 percentile602.0728.01056.0886.0
Max size2914.03409.03607.03607.0
Mean463.3262811408712542.6293830789272799.7239837957463662.6261153794035

rep1
rep1
rep2
rep2
idr_opt
idr_opt
overlap_opt
overlap_opt

Enrichment / Signal-to-noise ratio


TSS enrichment (filtered/deduped BAM)

rep1rep2
TSS enrichment23.29480544864944520.962438980086663

rep1
rep1
rep2
rep2

Open chromatin assays should show enrichment in open chromatin sites, such as TSS's. An average TSS enrichment in human (hg19) is above 6. A strong TSS enrichment is above 10. For other references please see https://www.encodeproject.org/atac-seq/


Jensen-Shannon distance (filtered/deduped BAM)

rep1rep2
AUC0.22634203991913450.24240738277199259
Synthetic AUC0.496269880977670830.4974896859719107
X-intercept0.105096483722120480.09602589426375682
Synthetic X-intercept0.00.0
Elbow Point0.70975313282205890.7516613753228786
Synthetic Elbow Point0.5018432465710.49871701474440583
Synthetic JS Distance0.39294827464987750.3810945307670934

Peak enrichment


Fraction of reads in peaks (FRiP)

FRiP for macs2 raw peaks

rep1rep2rep1-pr1rep2-pr1rep1-pr2rep2-pr2pooledpooled-pr1pooled-pr2
Fraction of Reads in Peaks0.30255057301012540.325571676944013340.295564607476114070.31768033911437530.30022286452655940.316529130531390370.32150496574722440.31550332148265080.3170344979769674

FRiP for overlap peaks

rep1_vs_rep2rep1-pr1_vs_rep1-pr2rep2-pr1_vs_rep2-pr2pooled-pr1_vs_pooled-pr2
Fraction of Reads in Peaks0.298342599176430.268565823632088360.307100272311501170.30705710411815457

FRiP for IDR peaks

rep1_vs_rep2rep1-pr1_vs_rep1-pr2rep2-pr1_vs_rep2-pr2pooled-pr1_vs_pooled-pr2
Fraction of Reads in Peaks0.249813659276828550.220272537534521780.26973701598628790.27180949470270777

For macs2 raw peaks:


For overlap/IDR peaks:

Other quality metrics


Comparison to Roadmap DNase

rep1
rep1
rep2
rep2

This bar chart shows the correlation between the Roadmap DNase samples to your sample, when the signal in the universal DNase peak region sets are compared. The closer the sample is in signal distribution in the regions to your sample, the higher the correlation.